The fields of biopharmaceutical processing and cell therapy are adopting single-use, closed systems throughout their workflows to enhance sterility, minimize waste wash effluent and enable manufacturing flexibility compared to traditional stainless steel bioreactors. One typical single-use technology in use is a bioreactor employing rocking motion, such as a WAVE Bioreactor™. A rocking bioreactor generally comprises a bag outfitted with ports and sensors, mounted on a tray, and a control system for controlling temperature, agitation, and media perfusion. The rocking motion of the bioreactor helps in mixing the contents of the bag during cell culture.
The cell culture bags employed for use in rocking bioreactors are made by sealing flat polymer films and the bags are stored flat until use. The bags are expanded upon filling with liquid growth medium and sparging gas during cell-culture. The medium and gas filled, expanded bags, made of polymer films, often develop creases and/or dimples, which can move under rocking motion. Over a period of time, the continuous imposition of cyclic stresses due to the rocking motion of the bioreactor, possibly in combination with the volumetric strain developed due to expansion of the medium filled bags and the motion of the liquid which reverse direction during each rocking cycle can lead to flexural fatigue of the polymer film used to manufacture the bags.
In the case of small-scale cell culture, generally, the cell culture bags are filled with lower volumes of media and the bags are subjected to less extreme rocking agitation. The low volume and/or lower rate of agitation results in deformation of the cell culture bag, but the deformation does not generally lead to the flexural fatigue and/or bag failure. However, the deformation in cell culture bags employed for rocking bioreactors can lead to flexural fatigue failures when the bags are used under extreme conditions, such as use of large media volume (such as, 25 liters or more), high rates of agitation generated from rocking motion of the bioreactor and/or long culture times (tens of days) or are improperly used (e.g., mis-mounted or underinflated). Flexural fatigue can cause a range of failures (e.g., whitening, delamination or cracking, followed by leakage and/or ingress of contaminants) of the cell culture bags used in rocking bioreactors, depending on a number of factors including the material and volume of the bag, internal pressure within the bag, angle at which the bioreactor is rocking, the rate of rocking of the bioreactor, and the duration of the rocking.
Several flexural fatigue-testing systems are currently available. However, none of these systems can effectively mimic the creases and/or dimples formed on a cell culture bag used in a rocking bioreactor, the geometry surrounding the creases and/or dimples on the bag, or the other forces imposed on the medium-filled bag during cell-culture and rocking of the bioreactor. The cell culture bags or films (used for such bags) are typically tested using a “rocker test”, which mimics the condition of using a rocking bioreactor on a film/bag and the test is essentially a real-time test of a film/bag used on a rocking bioreactor. However, while the rocker test can generate the dimples and/or creases on the bags, under conditions that the bags need to be used for large scale cell culture (e.g., at least 25 L scale), with continuous rocking at a high rate/angle for longer duration (e.g., more than a month), the rocker test for flexural fatigue testing is a low throughput, time consuming, labor intensive method, and it does not necessarily take films to failure (e.g., perforation or breaking) if a chosen time threshold is used.
There is thus a need for improved systems and methods, which can allow for rapid and reproducible flexural fatigue testing of the polymer film-based bags, which are typically used in bioreactors. Identification of fatigue resistant polymeric materials and/or bags made therefrom is a significant aspect of achieving robust cell cultures in bioreactor processes.